The mechanistic and transformative role of metabolism in tumor initiation and progression is a research topic of increasing importance. Isotopomer-based functional metabolomics and metabolic flux analysis are the most direct and informative approaches with which to study cellular metabolic processes. However, for a number of reasons, the robust isotopomer methodologies developed in bacterial systems are not commonly used in mammalian cancer research. This research proposal aims to evaluate and address the major issues confronting the application of isotopomer tracing in mammalian cells, including the use of non-glucose substrates, sample size and throughput, and the relevance of key tumor model systems. We will build on our recent development of a glucose-based metabolic model for human tumor cells to improve our detection capabilities, expand our isotopomer model and determine the extent of conservation between in vitro and in vivo models, all with the overall goal of facilitating the broader use of functional metabolomics in cancer research. This will directly support the both NCI's goal of funding research with a high potential for positive patient impact, and the IMAT program's goal of supporting the development of transformative technologies. Specifically, we are asking three interrelated questions. (1) How can we conserve the isotopic and chemical information provided by isotopic labeling while significantly decreasing sample size and increasing throughput? (2) Which metabolic fluxes can be quantified by tracking various metabolic substrates, and how is this information best incorporated into a comprehensive flux model? (3) To what extent does tumor cell metabolism in two-dimensional tissue culture reflect tumor metabolism in vivo? To answer these questions, we will (1) compare the isotopic information gained from GCMS analysis with that from NMR analysis and determine the how much information is conserved between the two methods;(2) perform a systematic evaluation of the utility of each of these compounds in mapping tumor cell metabolism in order to determine which metabolic pathways are most accessible to each precursor;and (3) compare the metabolic program of two-dimensional tissue culture, three-dimensional tissue culture and mouse xenograft models to determine which metabolic activities are conserved between systems and which vary with the microenvironmental conditions.